Our main scientific interest lies in developing conjugate vaccines for infectious diseases from carbohydrate antigens. Within these efforts, we have three ongoing projects. Two are concerned with a vaccine for cholera and one with a vaccine for anthrax. Existing vaccines for these diseases are based on cellular/bacterial material and, in addition to having undesirable side effects, they do not provide long-term protection. Development of vaccines for these two diseases is important from the point of view of public health and from the point of view of the national interest. The latter, because our armed forces are often active in geographical areas where these diseases are endemic. Anthrax does not constitute a major health problem in the civilized world. However, new concerns regarding anthrax have emerged because of a potential use of some form of Bacillus anthracis, which is the etiological cause of anthrax, as a biological weapon. The approach towards a vaccine for anthrax is based on preparation of neoglycoconjugates from a suitable carrier and the chemically synthesized tetrasaccharide side chain of the major glycoprotein of Bacillus anthracis exosporium. In the past, we have focused on improving diagnostic tools for the detection of the presence of anthrax spores. Since preliminary work indicated that the anthrose-containing tetrasaccharide chain seemed to be highly specific for B. anthracis, during the period associated with this report we focused on testing the possibility of altering immunogenicity of the protective antigen (PA), which is protein in nature, by coupling it with the tetrasaccharide moiety from the BclA protein. Preliminary immunization studies suggested that this tetrasaccharide construct might enhance the immune response generated by PA. This was indicated by approximately 20% increase of protection of mice challenged with Ames spores. Due to lack of funds on the side of our collaborators, further work on optimizing the conjugates was temporarily discontinued in the past period. Recently, the pharmaceutical company Novartis Vaccines in Siena, Italy, has shown interest in exploring possibilities to turn the tetrasaccharide side chain of the major glycoprotein of Bacillus anthracis exosporium synthesized in our laboratory into a vaccine for anthrax. Also, Novartis Vaccines is interested in looking at some immunochemical fundamentals concerning anthrax using synthetic fragments of the tetrasaccharide side chain of the major glycoprotein of Bacillus anthracis exosporium which we synthesized. Based on the Material Transfer Agreement we have established with Novartis Vaccines, we have provided the aforementioned materials to our Italian collaborators. Work towards those goals is ongoing, being conducted by the Novartis team, headed by Dr. Roberto Adamo, and a team of scientists at Uniform Services University. Our work towards a potent conjugate vaccine for cholera involves two approaches. In the first one, we synthesize oligosaccharides that mimic the structure of O-specific polysaccharide (O-PS) of Vibrio cholerae in the form suitable for conjugation, conjugate these antigens to suitable carriers, and serologically evaluate the immunogenicity of the resulting neoglycoconjugates. In the second approach, we chemically modify polysaccharides isolated from bacterial pathogens to make them amenable for conjugation, conjugate the resulting synthons to suitable protein carriers, and evaluate the utility of the newly formed constructs as experimental vaccines. We are expanding also the work based on our discovery we made in the past that bacterial O-SPcore antigens can be conjugated to proteins in the same, simple way as synthetic, linker-equipped carbohydrates by applying squaric acid chemistry. We have optimized our laboratory-scale protocol to make it useful for preparation of large batches of experimental vaccine from O-PScore of Vibrio cholerae O1 and a recombinant tetanus toxin fragment, and a protocol allowing large-scale preparation of cholera vaccine has already been developed by an outside of NIH contractor. In addition, we have completed academic explorations on the synthetic work towards the complete antigen of Vibrio cholerae O139, which is the prerequisite to making a vaccine from the fully synthetic vaccine for the disease caused by this bacterial pathogen. Also, exploratory work is ongoing towards a conjugate vaccine for other enteric diseases. Using synthetic Vibrio cholerae O1 hexasaccharide as an example, we demonstrated the use of a carbohydrate sub-carrier to synthesize a glycoconjugate where the carbohydrate antigen is displayed on the surface of the carrier protein in a clustered form. The immunoreactivities of the clustered and non-clustered conjugates were compared. While working on the above topics, we often digress, when scientifically interesting issues emerge, and exploit opportunities to answer fundamental questions relevant to conjugate vaccine development.